Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Discovering covalent inhibitors of protein–protein interactions from trillions of sulfur(VI) fluoride exchange-modified oligonucleotides

Nature Chemistry volume 15, pages 1705–1714 (2023)Cite this article

Subjects

Abstract

Molecules that covalently engage target proteins are widely used as activity-based probes and covalent drugs. The performance of these covalent inhibitors is, however, often compromised by the paradox of efficacy and risk, which demands a balance between reactivity and selectivity. The challenge is more evident when targeting protein–protein interactions owing to their low ligandability and undefined reactivity. Here we report sulfur(VI) fluoride exchange (SuFEx) in vitro selection, a general platform for high-throughput discovery of covalent inhibitors from trillions of SuFEx-modified oligonucleotides. With SuFEx in vitro selection, we identified covalent inhibitors that cross-link distinct residues of the SARS-CoV-2 spike protein at its protein–protein interaction interface with the human angiotensin-converting enzyme 2. A separate suite of covalent inhibitors was isolated for the human complement C5 protein. In both cases, we observed a clear disconnection between binding affinity and cross-linking reactivity, indicating that direct search for the aimed reactivity—as enabled by SuFEx in vitro selection—is vital for discovering covalent inhibitors of high selectivity and potency.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Strategy for SuFEx in vitro selection.
Fig. 2: Sequences that covalently modify the S1 subunit of the SARS-CoV-2 S protein discovered by SuFEx in vitro selection.
Fig. 3: Identification of cross-link sites in the RBD of the SARS-CoV-2 S protein.
Fig. 4: Reactivity and selectivity of truncated SF-Seq2 to the S1 subunit of the SARS-CoV-2 S protein.
Fig. 5: Neutralization of SARS-CoV-2 pseudoviruses by SF-Seq2-89-PEG.
Fig. 6: Covalent inhibitors of hC5 discovered by SuFEx in vitro selection.

Similar content being viewed by others

Data availability

The authors declare that data supporting the findings of this study are available within this article and the Supplementary information. The NGS raw data (fastq) for SuFEx in vitro selection are available from figshare via https://doi.org/10.6084/m9.figshare.23259986.v1 (ref. 55). The protein sequences and structures are accessible from Protein Data Bank (PDB), with entries 7A92, 6ZGI, 6ZGG and 6RQJ for S1/RBD, S protein trimer (close), S protein trimer (open) and hC5, respectively. Source Data are provided with this paper.

References

  1. Heal, W. P., Dang, T. H. T. & Tate, E. W. Activity-based probes: discovering new biology and new drug targets. Chem. Soc. Rev. 40, 246–257 (2011).

    Article  PubMed  CAS  Google Scholar 

  2. Wu, L. et al. An overview of activity-based probes for glycosidases. Curr. Opin. Chem. Biol. 53, 25–36 (2019).

    Article  PubMed  CAS  Google Scholar 

  3. Singh, J., Petter, R. C., Baillie, T. A. & Whitty, A. The resurgence of covalent drugs. Nat. Rev. Drug Discovery 10, 307–317 (2011).

    Article  PubMed  CAS  Google Scholar 

  4. Abdeldayem, A., Raouf, Y. S., Constantinescu, S. N., Moriggl, R. & Gunning, P. T. Advances in covalent kinase inhibitors. Chem. Soc. Rev. 49, 2617–2687 (2020).

    Article  PubMed  CAS  Google Scholar 

  5. Pettinger, J., Jones, K. & Cheeseman, M. D. Lysine-targeting covalent inhibitors. Angew. Chem. Int. Ed. 56, 15200–15209 (2017).

    Article  CAS  Google Scholar 

  6. Maurais, A. J. & Weerapana, E. Reactive-cysteine profiling for drug discovery. Curr. Opin. Chem. Biol. 50, 29–36 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  7. Narayanan, A. & Jones, L. H. Sulfonyl fluorides as privileged warheads in chemical biology. Chem. Sci. 6, 2650–2659 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  8. Lagoutte, R., Patouret, R. & Winssinger, N. Covalent inhibitors: an opportunity for rational target selectivity. Curr. Opin. Chem. Biol. 39, 54–63 (2017).

    Article  PubMed  CAS  Google Scholar 

  9. Moore, A. R., Rosenberg, S. C., McCormick, F. & Malek, S. RAS-targeted therapies: Is the undruggable drugged? Nat. Rev. Drug Discovery 19, 533–552 (2020).

    Article  PubMed  CAS  Google Scholar 

  10. Chen, S. et al. Identification of highly selective covalent inhibitors by phage display. Nat. Biotechnol. 39, 490–498 (2021).

    Article  PubMed  CAS  Google Scholar 

  11. Tivon, Y., Falcone, G. & Deiters, A. Protein labeling and crosslinking by covalent aptamers. Angew. Chem. Int. Ed. 60, 15899–15904 (2021).

    Article  CAS  Google Scholar 

  12. Sharpe, A. H. & Pauken, K. E. The diverse functions of the PD1 inhibitory pathway. Nat. Rev. Immunol. 18, 153–167 (2018).

    Article  PubMed  CAS  Google Scholar 

  13. Lan, J. et al. Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor. Nature 581, 215–220 (2020).

    Article  PubMed  CAS  Google Scholar 

  14. Ueda, T. et al. Enhanced suppression of a protein–protein interaction in cells using small-molecule covalent inhibitors based on an N-acyl-N-alkyl sulfonamide warhead. J. Am. Chem. Soc. 143, 4766–4774 (2021).

    Article  PubMed  CAS  Google Scholar 

  15. Gambini, L. et al. Covalent inhibitors of protein–protein interactions targeting lysine, tyrosine, or histidine residues. J. Med. Chem. 62, 5616–5627 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  16. Li, Q. K. et al. Developing covalent protein drugs via proximity-enabled reactive therapeutics. Cell 182, 85–97 (2020).

    Article  PubMed  CAS  Google Scholar 

  17. Han, Y. et al. Covalently engineered protein minibinders with enhanced neutralization efficacy against escaping SARS-CoV-2 variants. J. Am. Chem. Soc. 144, 5702–5707 (2022).

    Article  PubMed  CAS  Google Scholar 

  18. Wang, N. & Wang, L. Genetically encoding latent bioreactive amino acids and the development of covalent protein drugs. Curr. Opin. Chem. Biol. 66, 102106 (2022).

    Article  PubMed  CAS  Google Scholar 

  19. Tabuchi, Y., Yang, J. & Taki, M. Inhibition of thrombin activity by a covalent-binding aptamer and reversal by the complementary strand antidote. Chem. Commun. 57, 2483–2486 (2021).

    Article  CAS  Google Scholar 

  20. Cohen, M. S., Zhang, C., Shokat, K. M. & Taunton, J. Structural bioinformatics-based design of selective, irreversible kinase inhibitors. Science 308, 1318–1321 (2005).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  21. Bartel, D. P. & Szostak, J. W. Isolation of new ribozymes from a large pool of random sequences. Science 261, 1411–1418 (1993).

    Article  PubMed  CAS  Google Scholar 

  22. Chapman, K. B. & Szostak, J. W. In vitro selection of catalytic RNAs. Curr. Opin. Struct. Biol. 4, 618–622 (1994).

    Article  PubMed  CAS  Google Scholar 

  23. Liu, M., Chang, D. R. & Li, Y. F. Discovery and biosensing applications of diverse RNA-cleaving DNAzymes. Acc. Chem. Res. 50, 2273–2283 (2017).

    Article  PubMed  CAS  Google Scholar 

  24. McGhee, C. E., Loh, K. Y. & Lu, Y. DNAzyme sensors for detection of metal ions in the environment and imaging them in living cells. Curr. Opin. Biotechnol. 45, 191–201 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  25. Silverman, S. K. Pursuing DNA catalysts for protein modification. Acc. Chem. Res. 48, 1369–1379 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  26. Zhou, W. H., Saran, R. & Liu, J. W. Metal sensing by DNA. Chem. Rev. 117, 8272–8325 (2017).

    Article  PubMed  CAS  Google Scholar 

  27. Wang, Y. J., Nguyen, K., Spitale, R. C. & Chaput, J. C. A biologically stable DNAzyme that efficiently silences gene expression in cells. Nat. Chem. 13, 319–326 (2021).

    Article  PubMed  CAS  Google Scholar 

  28. Wang, Y. Y. et al. An RNA-cleaving threose nucleic acid enzyme capable of single point mutation discrimination. Nat. Chem. 14, 350–359 (2022).

    Article  PubMed  Google Scholar 

  29. Dunn, M. R., Jimenez, R. M. & Chaput, J. C. Analysis of aptamer discovery and technology. Nat. Rev. Chem. 1, 0076 (2017).

    Article  CAS  Google Scholar 

  30. Zhou, J. H. & Rossi, J. Aptamers as targeted therapeutics: current potential and challenges. Nat. Rev. Drug Discovery 16, 181–202 (2017).

    Article  PubMed  CAS  Google Scholar 

  31. Brighty, G. J. et al. Using sulfuramidimidoyl fluorides that undergo sulfur(VI) fluoride exchange for inverse drug discovery. Nat. Chem. 12, 906–913 (2020).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  32. He, Q., Johnston, J. & Zeitlinger, J. ChIP-nexus enables improved detection of in vivo transcription factor binding footprints. Nat. Biotechnol. 33, 395–401 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  33. Fidanza, J. A., Ozaki, H. & Mclaughlin, L. W. Site-specific labeling of DNA sequences containing phosphorothioate diesters. J. Am. Chem. Soc. 114, 5509–5517 (1992).

    Article  CAS  Google Scholar 

  34. Xiao, L., Gu, C. & Xiang, Y. Orthogonal activation of RNA-cleaving DNAzymes in live cells by reactive oxygen species. Angew. Chem. Int. Ed. 58, 14167–14172 (2019).

    Article  CAS  Google Scholar 

  35. Gu, C. M. et al. Chemical synthesis of stimuli-responsive guide RNA for conditional control of CRISPR-Cas9 gene editing. Chem. Sci. 12, 9934–9945 (2021).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  36. Dong, J. J., Krasnova, L., Finn, M. G. & Sharpless, K. B. Sulfur(VI) fluoride exchange (SuFEx): another good reaction for click chemistry. Angew. Chem. Int. Ed. 53, 9430–9448 (2014).

    Article  CAS  Google Scholar 

  37. Zheng, Q. H. et al. SuFEx-enabled, agnostic discovery of covalent inhibitors of human neutrophil elastase. Proc. Natl Acad. Sci. USA 116, 18808–18814 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  38. Morgan, B. P. & Harris, C. L. Complement, a target for therapy in inflammatory and degenerative diseases. Nat. Rev. Drug Discov. 14, 857–877 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  39. Hamady, M., Walker, J. J., Harris, J. K., Gold, N. J. & Knight, R. Error-correcting barcoded primers for pyrosequencing hundreds of samples in multiplex. Nat. Methods 5, 235–237 (2008).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  40. Liu, X. et al. Neutralizing aptamers block S/RBD-ACE2 interactions and prevent host cell infection. Angew. Chem. Int. Ed. 60, 10273–10278 (2021).

    Article  CAS  Google Scholar 

  41. Peinetti, A. S. et al. Direct detection of human adenovirus or SARS-CoV-2 with ability to inform infectivity using DNA aptamer-nanopore sensors. Sci. Adv. 7, eabh2848 (2021).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  42. Schmitz, A. et al. A SARS-CoV-2 spike binding DNA aptamer that inhibits pseudovirus infection by an RBD-independent mechanism. Angew. Chem. Int. Ed. 60, 10279–10285 (2021).

    Article  CAS  Google Scholar 

  43. Zhang, Z. et al. High-affinity dimeric aptamers enable the rapid electrochemical detection of wild-type and B.1.1.7 SARS-CoV-2 in unprocessed saliva. Angew. Chem. Int. Ed. 60, 24266–24274 (2021).

    Article  CAS  Google Scholar 

  44. Romaniuk, P. J. & Eckstein, F. A study of the mechanism of T4 DNA polymerase with diastereomeric phosphorothioate analogs of deoxyadenosine triphosphate. J. Biol. Chem. 257, 7684–7688 (1982).

    Article  PubMed  CAS  Google Scholar 

  45. Iwamoto, N. et al. Control of phosphorothioate stereochemistry substantially increases the efficacy of antisense oligonucleotides. Nat. Biotechnol. 35, 845–851 (2017).

    Article  PubMed  CAS  Google Scholar 

  46. Watanabe, Y., Allen, J. D., Wrapp, D., McLellan, J. S. & Crispin, M. Site-specific glycan analysis of the SARS-CoV-2 spike. Science 369, 330–333 (2020).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  47. Wrobel, A. G. et al. SARS-CoV-2 and bat RaTG13 spike glycoprotein structures inform on virus evolution and furin-cleavage effects. Nat. Struct. Mol. Biol. 27, 763–767 (2020).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  48. Xiong, X. L. et al. A thermostable, closed SARS-CoV-2 spike protein trimer. Nat. Struct. Mol. Biol. 27, 934–941 (2020).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  49. Cueno, M. E. & Imai, K. Structural comparison of the SARS-CoV-2 spike protein relative to other human-infecting coronaviruses. Front. Med. 7, 594439 (2020).

    Article  Google Scholar 

  50. Mukherjee, H. et al. A study of the reactivity of S(VI)-F containing warheads with nucleophilic amino-acid side chains under physiological conditions. Org. Biomol. Chem. 15, 9685–9695 (2017).

    Article  PubMed  CAS  Google Scholar 

  51. Nie, J. H. et al. Quantification of SARS-CoV-2 neutralizing antibody by a pseudotyped virus-based assay. Nat. Protoc. 15, 3699–3715 (2020).

    Article  PubMed  CAS  Google Scholar 

  52. Biesecker, G., Dihel, L., Enney, K. & Bendele, R. A. Derivation of RNA aptamer inhibitors of human complement C5. Immunopharmacology 42, 219–230 (1999).

    Article  PubMed  CAS  Google Scholar 

  53. Sefah, K. et al. In vitro selection with artificial expanded genetic information systems. Proc. Natl Acad. Sci. USA 111, 1449–1454 (2014).

    Article  PubMed  CAS  Google Scholar 

  54. Shangguan, D. et al. Aptamers evolved from live cells as effective molecular probes for cancer study. Proc. Natl. Acad. Sci. USA. 103, 11838–11843 (2006).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  55. Xiang, Y. NGS raw data of SuFEx in vitro selection. figshare https://doi.org/10.6084/m9.figshare.23259986.v1 (2023).

Download references

Acknowledgements

This work is supported by the National Key R&D Program of China (grant nos. 2021YFA1200900 to Y.X. and A.T., 2022YFE0102400 to Y.X., and 2018YFA0902601 to C.Z.), the National Natural Science Foundation of China (grant nos. 22074076 to Y.X., 21621003 to Y.X., 51973112 to C.Z., and 52225302 to C.Z.) and Tsinghua University Dushi Program (grant no. 20221080024 to Y.X.). We are grateful for the instrument assistance from the Analysis Center of Tsinghua University. We thank the support of all staffs from the Wuhan National Biosafety Level 3 Laboratory of Wuhan Institute of Virology, CAS. The figures in this article were created using BioRender (https://www.biorender.com/) and are licensed for publication.

Author information

Author notes

  1. Chunmei Gu

    Present address: Beijing Institute of Collaborative Innovation (BICI), Beijing, China

  2. These authors contributed equally: Zichen Qin, Kaining Zhang.

Authors and Affiliations

  1. Department of Chemistry, Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology (Ministry of Education), Tsinghua University, Beijing, China

    Zichen Qin, Kaining Zhang, Chunmei Gu, Yiying Zhu, Aijun Tong & Yu Xiang

  2. CAS Key Laboratory of Special Pathogens and Biosafety, Centre for Biosafety Mega-Science, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China

    Ping He & Hongping Wei

  3. School of Chemistry and Chemical Engineering, Frontiers Science Center for Transformative Molecules, Shanghai Key Laboratory for Molecular Engineering of Chiral Drugs, State Key Laboratory of Metal Matrix Composites, Shanghai Jiao Tong University, Shanghai, China

    Xue Zhang, Miao Xie & Chuan Zhang

  4. Department of Orthopedics, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China

    Yucheng Fu

Authors
  1. Zichen Qin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kaining Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ping He
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Xue Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Miao Xie
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Yucheng Fu
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Chunmei Gu
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Yiying Zhu
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Aijun Tong
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Hongping Wei
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Chuan Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Yu Xiang
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Z.Q., K.Z., C.G., A.T. and Y.X. conceived and designed the SuFEx in vitro selection. Z.Q. and Y.X. performed S1 SuFEx in vitro selection. Z.Q. and K.Z. performed RBD and hC5 SuFEx in vitro selection. Z.Q. and K.Z. synthesized the SuFEx-modified oligonucleotides, performed the PAGE characterization and analysed the results. Y.Z. performed LC–MS/MS measurements and analysed the results for cross-linking site assignment. P.H. and H.W. designed and performed the SARS-CoV-2 neutralization assays, and analysed the results. Z.Q. and Y.X. designed and performed the pseudovirus neutralization assays, and analysed the results. X.Z., M.X., Y.F. and C.Z. designed and performed the hC5-induced haemolysis inhibition assays, and analysed the results. Z.Q., K.Z. and Y.X. wrote the paper. All authors discussed the results and commented on the paper. Z.Q. and K.Z. contributed equally to this work.

Corresponding author

Correspondence to Yu Xiang.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Chemistry thanks Jiajia Dong, Seiya Kitamura and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figs. 1–22 and Tables 1–3.

Reporting Summary

Supplementary Data 1

Oligonucleotide sequences used.

Supplementary Data 2

Raw data for plots in Supplementary Information.

Supplementary Data 3

NGS reads for all selections.

Source data

Source Data Fig. 2

Uncropped gels in Fig. 2c–e.

Source Data Fig. 4

Uncropped gels in Fig. 4b–f.

Source Data Fig. 5

Raw numerical/statistical data in Fig. 5b,d,e.

Source Data Fig. 5

The raw fluorescence and bright field images in Fig. 5c.

Source Data Fig. 6

Uncropped gels in Fig. 6c,d.

Source Data Fig. 6

Raw numerical/statistical data in Fig. 6e.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Qin, Z., Zhang, K., He, P. et al. Discovering covalent inhibitors of protein–protein interactions from trillions of sulfur(VI) fluoride exchange-modified oligonucleotides. Nat. Chem. 15, 1705–1714 (2023). https://doi.org/10.1038/s41557-023-01304-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41557-023-01304-z

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing